Receptor for B anthracis toxin

ABSTRACT

The present invention relates to mammalian anthrax toxin receptor polypeptides and polynucleotides encoding same as well as related polypeptides and polynucleotides, vectors containing the polynucleotides and polypeptides, host cells containing related polynucleotide molecules, and cells displaying no anthrax toxin receptor on an exterior surface of the cells-minus cell lines and animals. The present invention also relates to methods for identifying molecules that bind the anthrax toxin receptor and molecules that reduce the toxicity of anthrax toxin. Finally, the present invention provides methods for treating human and non-human animals suffering from anthrax.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. provisional application Ser.No. 60/251,481, filed on Dec. 5, 2000, which is incorporated herein byreference as if set forth in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with United States government support awarded bythe following agencies:

-   -   NIH AI48489 and NIH AI22021.

The United States has certain rights in this invention.

BACKGROUND OF THE INVENTION

Bacillus anthracis, the spore-forming causative agent of anthrax,generally infects herbivores (Hanna, 1998). Human infection, while rare,can result in a generally benign, self-limiting cutaneous disease or asystemic disease that rapidly leads to death in a high percentage ofcases. The cutaneous disease can arise when spore particles from soil oranimal products are introduced into cuts or skin abrasions. In contrast,the systemic disease can arise when B. anthracis spore particles areinhaled (LD₅₀ ≈10,000 spore particles). The high mortality rate and theability to readily prepare and deliver B. anthracis spore particles asan aerosol have made B. anthracis a dreaded agent of biowarfare andbioterrorism.

The causative agent of the systemic disease is anthrax toxin (AT), whichitself comprises a pair of binary, AB-type toxins—lethal toxin and edematoxin (Leppla, 1995). Each is assembled at the surface of mammaliancells from proteins released by B. anthracis. Lethal toxin, assembledfrom Protective Antigen (PA, 83 kDa) and Lethal Factor (LF, 90 kDa), isprimarily responsible for lethality (Friedlander, 1986; Hanna et al.,1992; Hanna et al., 1993). Edema toxin, assembled from PA and EdemaFactor (EF, 89 kDa), causes edema at the site of injection (Leppla,1982). EF has calmodulin-dependent adenylate cyclase activity. LF is aZn⁺⁺-dependent protease that cleaves certain proteins involved in signaltransduction and cell cycle progression (MAPKK1 and MAPKK2) (Duesbery etal., 1998).

In these AB-type toxins, PA is the receptor-binding B moiety thatdelivers either EF or LF, as alternative enzymic A moieties, to thecytosol of mammalian cells (Leppla, 1995). Initially, PA bindsspecifically, reversibly, and with high affinity (Kd≈1 nM) to acell-surface AT receptor (ATR). After binding to the receptor, PA iscleaved by a member of the furin family of proprotein convertases, whichremoves a 20 kDa fragment, PA20, from the N-terminus (Klimpel et al.,1992; Novak et al., 1992). The complementary fragment, PA63, remainsreceptor-bound and spontaneously self-associates to form heptamericring-shaped oligomers (Milne et al., 1994) that avidly and competitivelybind EF and/or LF (Leppla, 1995) to form EF/LF-PA63 complexes. Thesecomplexes are trafficked to an acidic compartment by receptor-mediatedendocytosis. In the acidic compartment, the PA63 heptamers (the“prepore”) are inserted into the membrane, forming transmembrane pores(Gordon et al., 1988). Concomitantly EF and LF are translocated acrossthe membrane to the cytosol. Consistent with the pH dependence oftranslocation, toxin action is inhibited by lysosomotropic agents andbafilomycin A1 (Mendard et al., 1996).

EF translocation causes a large increase in intracellular cAMPconcentration (Gordon et al., 1988; Gordon et al., 1989). Increased cAMPlevels cause edema, and in neutrophils, inhibit phagocytosis andoxidative burst (O'Brien et al., 1985). By protecting the bacteria fromphagocytosis, edema toxin apparently aids in establishing bacterialinfection and proliferation in the mammalian host.

Treatment of primary macrophages and certain macrophage cell lines withlethal toxin causes cell lysis (Friedlander, 1986). Macrophage-depletedmice are resistant to treatment with lethal toxin, suggesting thatmacrophages are the primary targets of lethal toxin (Hanna et al.,1993). Low doses of lethal toxin induce the production of interleukin-1and tumor necrosis factor (Hanna et al., 1993). Thus, it has beensuggested that hyperproduction of cytokines causes death of the host byinducing systemic shock. How these or other proteins lead to cytokineproduction and macrophage lysis remains unclear.

In the past few years considerable progress has been made toward adetailed understanding of the structure and function of PA.Crystallographic structures of PA and the PA63 heptamers have beendetermined (Petosa et al., 1997). The prepore undergoes a majorconformational change under acidic conditions to form a 14-strandtransmembrane β-barrel pore (Benson et al., 1998; Miller et al., 1999).The pore structure and the detailed mechanism by which LF and EF aretranslocated across membranes are under intensive investigation.

The ATR structure is heretofore unknown, but is present in all celllines that have been tested. Studies on CHO-K1 cells had indicated thatPA binds to a proteinaceous receptor that is present in about 10⁴copies/cell (Escuyer and Collier, 1991). The paucity of knowledge aboutthe ATR represents a major gap in the understanding of how AT acts.Identification and cloning of the ATR will provide more treatmentstrategies for anthrax.

A cDNA clone (Genbank Accession Number NM 032208) known as tumorendothelial marker 8 (TEM8) is known (St. Croix, 2000). TEM8 isupregulated in colorectal cancer endothelium, but heretofore thefunction of TEM8 was not known.

BRIEF SUMMARY OF THE INVENTION

The present application discloses structures of complete and partialanthrax toxin receptors from a mammal, namely a human. The completeanthrax toxin receptor includes an extracellular domain, a transmembranedomain, and a cytoplasmic domain that can vary in length, as isdisclosed herein. It is disclosed herein that PA binds to the anthraxtoxin receptor at a von Willebrand factor A (VWA) domain in theextracellular domain.

In one aspect, the invention is summarized in that an anthrax toxinreceptor is a polypeptide having an amino acid sequence selected fromSEQ ID NO:2, SEQ ID NO:6, SEQ ID NO: 8, SEQ ID NO:10, a PA-bindingfragment of any of the foregoing, and a PA-binding variant of any of theforegoing polypeptides having conservative or non-conservative aminoacid substitutions or other changes relative to the disclosed sequences.The various forms of the receptor encoded by SEQ ID NO:2, SEQ ID NO:6,SEQ ID NO: 8, and SEQ ID NO:10 apparently differ as a result ofalternative splicing.

In a related aspect, the invention further relates to an isolatedpolynucleotide that encodes any of the above-mentioned polypeptides andtheir complements, and a polynucleotide that hybridizes under moderatelystringent or stringent hybridization conditions to any of the foregoing.

In still another related aspect, the invention encompasses a cloningvector and an expression vector comprising any of the foregoingpolynucleotides, whether or not the polynucleotide is operably linked toan expression control sequence that does not natively promotetranscription or translation of the polynucleotide.

By identifying the polypeptides and polynucleotides of the invention,the applicant enables the skilled artisan to detect and quantify mRNAand ATR protein in a sample, and to generate atr transgenic and atrknock-out animals using methods available to the art.

Further, the invention includes a host cell comprising any such vectorin its interior. Also within the scope of the present invention is ahost cell having a polynucleotide of the invention integrated into thehost cell genome at a location that is not the native location of thepolynucleotide.

In yet another aspect, the invention is a method for producing ananthrax toxin receptor polypeptide that includes the steps oftranscribing a polynucleotide that encodes an anthrax toxin receptorpolypeptide, operably linked to an upstream expression control sequence,to produce an mRNA for the receptor polypeptide, and translating themRNA to produce the receptor polypeptide. This method can be performedin a host cell when the polynucleotide is operably linked to theexpression control sequence in an expression vector, and wherein theexpression vector is delivered into a host cell, the expression controlsequence being operable in the host cell. Alternatively, at least one ofthe transcribing and translating steps can be performed in an in vitrosystem, examples of which are well known in the art and commerciallyavailable. In either case, the polypeptide can be isolated from othercellular material using readily available methods.

In still another aspect, the invention is a method for identifying anagent that can alter the effect of AT on the host cell or organism. Themethod includes the steps of separately exposing a plurality of putativeagents in the presence of AT to a plurality of cells having on theirsurface at least a portion of the ATR that binds to AT or a componentthereof, comparing the effect of AT on the cells in the presence andabsence of the agent, and identifying at least one agent that alters aneffect of AT on the cells. In a related aspect, the present inventionencompasses an agent that alters binding of AT to the ATR.

The present invention also encompasses a method for reducing orpreventing AT-related damage in vivo or in vitro to human or non-humancells having an ATR on an outer cell surface, the method comprising thestep of exposing the cells to an agent that reduces binding of AT to theATR. Similarly, the invention relates to a method for reducing orpreventing damage in vivo or in vitro to human or non-human cells causedby AT by exposing AT to an agent that reduces binding of the AT to theATR.

The present invention is also a method for identifying a mutant of theextracellular ATR domain or fragment thereof having altered (increasedor reduced) binding affinity for AT.

It is an object of the invention to identify polypeptides that encode amammalian anthrax toxin receptor, as well as fragments, mutants, andvariants thereof and polynucleotides encoding same.

It is a feature of the invention that a soluble PA-binding polypeptidecan reduce or eliminate toxicity associated with anthrax toxin.

Other objects, advantages and features of the invention will becomeapparent from the following specifications and claims.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

FIG. 1 shows sequence alignment of various ATR polypeptide sequenceswith the I domain of integrin α2 and with the von Willebrand factor Adomain consensus sequence.

DETAILED DESCRIPTION OF THE INVENTION

An isolated polynucleotide and an isolated polypeptide, as used herein,can be isolated from its natural environment or can be synthesized.Complete purification is not required in either case. Amino acid andnucleotide sequences flanking an isolated polypeptide or polynucleotidethat occurs in nature, respectively, can but need not be absent from theisolated form.

Further, an isolated polynucleotide has a structure that is notidentical to that of any naturally occurring nucleic acid or to that ofany fragment of a naturally occurring genomic nucleic acid spanning morethan three separate genes. The term includes, without limitation, (a) anucleic acid molecule having a sequence of a naturally occurring genomicor extrachromosomal nucleic acid molecule but which is not flanked bythe coding sequences that flank the sequence in its natural position;(b) a nucleic acid molecule incorporated into a vector or into aprokaryote or eukaryote genome such that the resulting molecule is notidentical to any naturally occurring vector or genomic DNA; (c) aseparate molecule such as a cDNA, a genomic fragment, a fragmentproduced by polymerase chain reaction (PCR), or a restriction fragment;and (d) a recombinant nucleotide sequence that is part of a hybrid gene,i.e., a gene encoding a fusion protein. Specifically excluded from thisdefinition are nucleic acids present in mixtures of clones, e.g., asthese occur in a DNA library such as a cDNA or genomic DNA library. Anisolated nucleic acid molecule can be modified or unmodified DNA or RNA,whether fully or partially single-stranded or double-stranded or eventriple-stranded. A nucleic acid molecule can be chemically orenzymatically modified and can include so-called non-standard bases suchas inosine.

Reference herein to use of AT is understood to encompass use of anATR-binding component thereof, especially PA.

Anthrax Toxin Receptor

The applicants have identified and determined the nucleic acid sequence(SEQ ID NO:1) of a cDNA clone that of a 368 amino acid long polypeptide(SEQ ID NO:2, ATR), and show herein that the polypeptide is asurface-bound anthrax toxin receptor (ATR) on human cells. Based onknown structural analysis methods, the polypeptide is predicted toencode a 27 amino-acid-long signal peptide (amino acids 1–27 of SEQ IDNO:2), a 293 amino-acid-long extracellular domain (amino acids 28–320 ofSEQ ID NO:2), a 23 amino-acid-long putative transmembrane region (aminoacids 320–343 of SEQ ID NO:2), and a 25 amino acid long cytoplasmicdomain (amino acids 344–368 of SEQ ID NO:2).

It is disclosed herein that Protective Antigen (PA) of anthrax toxin(AT) binds to the anthrax toxin receptor at a von Willebrand factor A(VWA) domain located in the portion from amino acid 44 to 216 in theextracellular domain of SEQ ID NO:2. VWA domains are present in theextracellular portions of a variety of cell surface proteins, includingmatrilins and integrins (designated as I domains). A VWA domainconsensus sequence, VWA-CON, developed by comparing 210 relatedsequences, is presented as SEQ ID NO:3. These domains are important forprotein/protein interactions and constitute ligand binding sites forintegrins (Dickeson, 1998). The I domain of integrin α2 (α2) ispresented as SEQ ID NO:4. Ligand binding through I domains requires anintact metal ion-dependent adhesion site (MIDAS) motif (Lee, 1995) whichappears to be conserved in the ATR extracellular domain, as is detailedbelow.

Comparison of SEQ ID NO:1 and SEQ ID NO:2 to existing databases revealedother versions of those sequences. Human cDNA TEM8 (SEQ ID NO:5; Genbankaccession number NM 032208) encodes a 564 amino-acid-long form (SEQ IDNO:6) of the human ATR. SEQ ID NO:6 has not previously been identifiedas an anthrax toxin receptor, and indeed no function has yet beenascribed to the protein. Like SEQ ID NO:1, SEQ ID NO:5 was a PCRamplification product from HeLa cells and human placenta cDNA libraries.Whereas the cytoplasmic tail of SEQ ID NO:2 is only 25 amino acids long,that of SEQ ID NO:6 is predicted to be 221 amino acids long (amino acids344–564), presumably as a result of differential splicing of a primarymRNA transcript. The proteins are otherwise identical. Upstream of thecoding sequences, SEQ ID NO:1 and SEQ ID NO:5 are also identical.

Also presented are IMAGE CLONE 4563020 (SEQ ID NO:7; Genbank AccessionNumber BC012074) and the predicted polypeptide encoded by the clone (SEQID NO:8). SEQ ID NO:8 is identical to amino acids 1–317 of ATR, butdiffers thereafter at the C-terminus. Similarly, human cDNA FLJ10601,clone NT2RP2005000 (SEQ ID NO:9; Genbank Accession Number AK001463) andthe predicted polypeptide encoded by the clone (SEQ ID NO:10) arepresented. This polypeptide is identical to a portion of SEQ ID NO:2from amino acid 80 to amino acid 218. As with TEM8 and the protein itencodes, no function is known for any of these polynucleotide andpolypeptide sequences, nor has there been any prior indication that thepolypeptides are complete or partial anthrax toxin receptors.

It is of interest to note that the product of the mouse homolog ofATR/TEM8 (Genbank accession number AK013005) is highly related to thehuman clones, sharing greater than 98% amino acid sequence identitywithin the reported extracellular domain. This suggests that the anthraxtoxin receptor is conserved among species. Furthermore, consistent withthe observation that the anthrax toxin receptor is found in a variety ofcell lines, ATR is expressed in a number of different tissues includingCNS, heart, lung, and lymphocytes.

In addition to the full-length and partial ATR polypeptide sequencespresented in SEQ ID NO:2, SEQ ID NO:6, SEQ ID NO:8 and SEQ ID NO:10,other polypeptide fragments shorter than those sequences that retainPA-binding activity, and variants thereof are also within the scope ofthe invention. The entire receptor is not required for utility; rather,fragments that bind to PA are useful in the invention.

A skilled artisan can readily assess whether a fragment binds to PA. Apolypeptide is considered to bind to PA if the equilibrium dissociationconstant of the binary complex is 10 micromolar or less. PA-binding tothe ATR (or a fragment of the ATR) can be measured using aprotein—protein binding method such as coimmunoprecipitation, affinitycolumn analysis, ELISA analysis, flow cytometry or fluorescenceresonance energy transfer (FRET), and surface plasmon resonance (SPR).SPR is particularly suited as it is highly sensitive and accurate,operable in real time, and consumes only minute amounts of protein. SPRuses changes in refractive index to quantify macromolecular binding anddissociation to a ligand covalently tethered to a thin gold chip in amicro flow cell. Besides the equilibrium dissociation constant (Kd), on-and off-rate constants (ka and kd) can also be obtained. A BIAcore 2000instrument (Pharmacia Biotech) can be used for these measurements.Typically, a protein is covalently tethered to a carboxymethyl dextranmatrix bonded to the gold chip. Binding of a proteinaceous ligand to theimmobilized protein results in a quantifiable change in refractive indexof the dextran/protein layer. SPR can also be used to determine whetherthe interaction between PA and its receptor is sensitive to low pH,which is relevant to toxin endocytosis. This technique has been used tostudy protein—protein interactions in many systems, including theinteractions of PA63 with EF and LF (Elliott, 1998).

The invention also relates to polypeptides that are at least 80%,preferably at least 90%, more preferably at least 95%, still morepreferably at least 97%, or most preferably at least 99% identical toany aforementioned PA-binding polypeptide fragment, where PA-binding ismaintained. As used herein, “percent identity” between amino acid ornucleic acid sequences is synonymous with “percent homology,” which canbe determined using the algorithm of Karlin and Altschul (Proc. Natl.Acad. Sci. USA 87:2264–2268, 1990), modified by Karlin and Altschul(Proc. Natl. Acad. Sci. USA 90:5873–5877, 1993). Such an algorithm isincorporated into the NBLAST and XBLAST programs of Altschul et al. (J.Mol. Biol. 215:403–410, 1990). BLAST nucleotide searches are performedwith the NBLAST program, score=100, wordlength=12, to obtain nucleotidesequences homologous to a nucleic acid molecule of the invention. BLASTprotein searches are performed with the XBLAST program, score=50,wordlength=3, to obtain amino acid sequences homologous to a referencepolypeptide (e.g., SEQ ID NO:2). To obtain gapped alignments forcomparison purposes, Gapped BLAST is utilized as described in Altschulet al. (Nucleic Acids Res. 25:3389–3402, 1997). When utilizing BLAST andGapped BLAST programs, the default parameters of the respective programs(e.g., XBLAST and NBLAST) are used. The referenced programs areavailable on line from the National Center for BiotechnologyInformation, National Library of Medicine, National Institute of Health.A variant can also include, e.g., an internal deletion or insertion, aconservative or non-conservative substitution, or a combination of thesevariations from the sequence presented.

Soluble fragments are of great interest as these can competitivelyinhibit anthrax toxin binding to the ATR and thereby can protect cellsfrom AT intoxication in vivo and in vitro. A fragment is soluble if itis not membrane-bound and is soluble in an aqueous fluid. Theextracellular ATR domain is a soluble fragment of the ATR, as arefragments of that domain. Even though the VWA domain is formallyidentified as extending from amino acid 44 to 216 in the extracellulardomain, more or fewer natively adjacent amino acids can be included inthe fragment without compromising solubility or PA-binding. For example,a PA-binding fragment having the sequence of SEQ ID NO:2 beginning atany amino acid in the range from 27 to 43 and ending at any amino acidin the range from 221 to 321. A preferred soluble, PA-binding fragmentextends from amino acid 42 to 222. Another preferred soluble PA-bindingfragment includes a fragment of the ATR from amino acid 27 through aminoacid 321. Likewise, any polypeptide fragment of these preferredfragments that retains PA-binding activity is within the scope of theinvention. ATR in soluble form is effective in a monomeric form, as wellas in multimeric forms such as dimeric, tetrameric, pentameric andhigher oligomeric forms.

PA-binding polypeptides can include, therefore, SEQ ID NO:2, SEQ IDNO:6, SEQ ID NO:8, SEQ ID NO:10, a PA-binding fragment of SEQ ID NO:2, aPA-binding fragment of SEQ ID NO:6, a PA-binding fragment of SEQ IDNO:8, a PA-binding fragment of SEQ ID NO:10, a PA-binding polypeptide atleast 80% identical to any of the foregoing fragments. The PA-bindingpolypeptides can also be provided as fusion proteins comprising any ofthe foregoing that can comprise still other non-natively adjacent aminoacids for detecting, visualizing, isolating, or stabilizing thepolypeptide. For example, PA binds to a soluble fusion protein of ahexahistidine tag, a T7 tag, and amino acids 41–227 of ATR.

Likewise, isolated polynucleotides having an uninterrupted nucleic acidsequence that encodes the aforementioned polypeptides and polypeptidefragments are also useful in the invention. The sequences that encodesoluble, PA-binding polypeptide fragments of ATR are immediatelyapparent to the skilled artisan from the description of the relevantportions of the polypeptides, supra. An isolated nucleic acid containingthe complement of any such polynucleotide is also within the scope ofthe present invention, as are polynucleotide and oligonucleotidefragments for use as molecular probes. The polynucleotides of theinvention cannot encode SEQ ID NO:6, SEQ ID NO:8 or SEQ ID NO:10.

The present invention also relates to an isolated polynucleotide and itscomplement, without regard to source, where the polynucleotidehybridizes under stringent or moderately stringent hybridizationconditions to SEQ ID NO:1, SEQ ID NO:5, SEQ ID 7, or SEQ ID NO:9 or to afragment of any of the foregoing that encodes a soluble polypeptide thatcan bind to PA. As used herein, stringent conditions involve hybridizingat 68° C. in 5×SSC/5× Denhardt's solution/1.0% SDS, and washing in0.2×SSC/0.1% SDS+/−100 μg/ml denatured salmon sperm DNA, at roomtemperature. Moderately stringent conditions include washing in the samebuffer at 42° C. Additional guidance regarding such conditions isreadily available in the art, for example, by Sambrook et al., 1989,Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, N.Y.;and Ausubel et al. (eds.), 1995, Current Protocols in Molecular Biology,(John Wiley & Sons, N.Y.) at Unit 2.10.

In a related aspect, any polynucleotide of the invention can be providedin a vector in a manner known to those skilled in the art. The vectorcan be a cloning vector or an expression vector. In an expressionvector, the polypeptide-encoding polynucleotide is under thetranscriptional control of one or more non-native expression controlsequences, such as a promoter not natively adjacent to thepolynucleotide, such that the encoded polypeptide can be produced whenthe vector is delivered into a compatible host cell that supportsexpression of an polypeptide encoded on a vector, for example byelectroporation or transfection, or transcribed and translated in acell-free transcription and translation system. Such cell-based andcell-free systems are well known to the skilled artisan. Cellscomprising an insert-containing vector of the invention are themselveswithin the scope of the present invention, without regard to whether thevector is extrachromosomal or integrated in the genome.

A skilled artisan in possession of the polypeptides and polynucleotidesof the invention can also identify agents that can reduce or prevent theeffect of AT on a host having on the cell surface at least a portion ofthe ATR. The effect altered can relate, for example, to (1)susceptibility of the host cell to AT damage, (2) integration of ATRinto the cell membrane, (3) binding between ATR and PA, (4) PAheptamerization, (5) uptake of PA and ATR complex into cells, and (6)the translocation of toxin into host cell cytoplasm. The method includesseparately exposing a plurality of putative agents in the presence of ATto a plurality of cells, comparing the effect of AT on the cells in thepresence and absence of the agent, and identifying at least one agentthat alters an effect of AT on the cells.

The skilled artisan can readily evaluate the typical effects of AT andcan observe variations in those effects in the presence of a putativealtering agent. For example, susceptibility to AT damage can beevaluated by exposing host cells to AT. Integration of newly formed ATRinto the host cell membrane can be evaluated by labeling newlysynthesized proteins in the host cell and immunopreticipating ATR fromthe cellular membrane fraction of the host cell. Binding of wild-typeATR to PA can be evaluated with fluorescent labeled anti-PA antibody. PAheptamerization can be evaluated by several techniques including nativepolyacrylamide gel electrophoresis, gel filtration, and westernblotting. Uptake of PA-ATR complex can be evaluated by binding PA to ATRat 4° C., increasing the temperature to 37° C. to allow endocytosis,shifting the temperature back to 4° C., and incubating cells withfluorescent labeled anti-PA antibodies. Toxin translocation into thehost cell cytoplasm can be evaluated as described in Wesche et al, 1998,which is incorporated herein by reference as if set forth in itsentirety.

The agents screened can be, for example, dominant negative mutant ATRs(encoded by a mutant polynucleotide sequence, which can be provided inan expression vector), a high molecular weight molecule such as apolypeptide (including, e.g., a mutant AT, a soluble ATR, a mono- orpolyclonal antibody to an ATR, to PA, or to an ATR/PA complex), apolysaccharide, a lipid, a nucleic acid, a low molecular weight organicor inorganic molecule, or the like. Antibodies can be produced byadministering to a non-human animal an immunogenic, PA-binding fragmentof a polypeptide which can be, e.g., SEQ ID NO:2, SEQ ID NO:6, SEQ IDNO:8, SEQ ID NO:10, a polypeptide at least 80% identical to any of theforegoing and a fusion protein comprising any of the foregoing, and thenobtaining the desired antibodies using known methods.

Chemical libraries for screening putative agents, including peptidelibraries, are readily available to the skilled artisan. Examplesinclude those from ASINEX (i.e. the Combined Wisdom Library of 24,000manually synthesized organic molecules) and from CHEMBRIDGE CORPORATION(i.e. the DIVERSet™ library of 50,000 manually synthesized chemicalcompounds; the SCREEN-Set™ library of 24,000 manually synthesizedchemical compounds; the CNS-Set™ library of 11,000 compounds; theCherry-Pick™ library of up to 300,000 compounds) and linear library,multimeric library and cyclic library (Tecnogen (Italy)). Once an agentwith desired activity is identified, a library of derivatives of thatagent can be screened for better agents. Phage display is also asuitable approach for finding novel inhibitors of the interactionbetween PA and ATR.

Another aspect of the present invention relates to ATR ligands otherthan PA and methods for identifying ATR ligands. As ATR is expressed inmany cell types, it likely has other natural ligands. To identify theseother ligands, a polypeptide that contains an ATR VWA domain, preferablyan entire extracellular domain can be provided in soluble or tetheredform, e.g., in a chromatographic column. Preferably, the ectodomain ofATR can be provided as a fusion protein that also a contains rabbit IgGconstant region, a GST domain or a hexahistidine tag. This fusionprotein can be immobilized on a chromatographic column using knownmethods. A cell extract can be passed over the column. A ligand isidentified when binding is observed between the ectodomain and acompound present in the cell extract. The identified ligand can be usedin methods for identifying agents that alter an effect of AT, toidentify an agent that selectively inhibits PA-ATR binding. It is alsodesirable to use the other ligands and the ATR in comparative highthroughput screening methods for identifying small molecules that do notinterfere with natural ligand binding to ATR, but which do prevent orreduce binding of ATR to anthrax toxin.

The present invention also relates to reducing cellular damage caused byAT, which can be achieved by administering an agent for reducing the ATRlevel, inhibiting the binding between ATR and AT, or by reducingdownstream ATR activity after AT binding. For example, an antisenseoligonucleotide can reduce or prevent expression of atr using deliverymethods known to the skilled artisan, thus reducing the cellular ATRlevel. An ATR-anthrax binding inhibition agent can inhibit the bindingbetween ATR and AT. Dominant negative ATRs can block downstream ATRactivities required for AT toxicity. The agents used for reducing ATdamage to cells can be administered to a human or non-human animal,preferably in a standard pharmaceutical carrier, in an amount effectiveto reduce or eliminate anthrax toxicity.

A 20–25mer antisense oligonucleotide can be directed against 5′ end ofthe atr message with phosphorothioate derivatives on the last three basepairs on the 3′ end and the 5′ end to enhance the half life andstability of the oligonucleotides. A carrier for an antisenseoligonucleotide can be used. An example of a suitable carrier iscationic liposomes. For example, an oligonucleotide can be mixed withcationic liposomes prepared by mixing 1-alphadioleylphatidylcelthanolamine with dimethldioctadecylammonium bromide ina ratio of 5:2 in 1 ml of chloroform. The solvent will be evaporated andthe lipids resuspended by sonication in 10 ml of saline. Another way touse an antisense oligonucleotide is to engineer it into a vector so thatthe vector can produce an antisense cRNA that blocks the translation ofthe mRNAs encoding for ATR. Similarly, RNAi techniques, which are nowbeing applied to mammalian systems, are also suited for inhibiting ATRexpression (see Zamore, Nat. Struct. Biol. 8:746:750 (2001),incorporated herein by reference as if set forth in its entirety).

The present invention also relates to a method for detecting atr mRNA orATR protein in a sample. Such detection can be readily accomplished byusing oligonucleotide or polynucleotide probes for atr mRNA, orantibodies for ATR protein. In a related aspect, the antibodies made andidentified as being able to bind to ATR can also be used to separate ATRfrom a sample.

The present invention also relates to a cell line that does not containATR from a parent cell line that contains ATR, and methods for makingsame. The present invention provides that it is possible for cellslacking ATR to survive. In the example described below, a cell line thatdoes not contain ATR was created using mutagenesis and screening. Nowthat the atr cDNA sequence is identified in the present invention, manyother methods for generating a cell line that does not express atrbecome feasible, such as homologous recombination. In addition to thesemethods, the cell lines generated, including the one described in theexample below, are themselves within the scope of the present invention.

The invention also provides molecules and methods for specificallytargeting and killing cells of interest by delivering, e.g., AT or LF tothe cell. Soluble ATR molecules can be coupled to a ligand or to asingle chain antibody selected for targeting to the cell of interest(e.g., a ligand that binds a receptor presented on a tumor cellsurface). The coupling is most readily accomplished by producing afusion protein that encodes both the ATR binding portion and the ligandor single chain antibody molecule. The ligand or single chain antibodydomains simply serve to attach the toxin to cells with the cognatesurface markers. The toxin or factor is preloaded onto the ATR portionbefore exposing the coupled molecules to the targeted cells. This issimilar in principle to the previously described for retroviraltargeting using soluble retroviral receptor-ligand bridge proteins andretroviral receptor-single chain antibody bridge proteins. SeeSnitkovsky and Young, Proc. Natl. Acad. Sci. USA 95:7063–7068 (1998);Boerger et al. Proc. Natl. Acad. Sci. USA 96:9687–9872 (1999) andSnitkovsky et al., J. Virol. 74:9540–9545 (2000), and Snitkovsky et al.,J. Virol. 75:1571–1575 (2001), each incorporated herein by reference asif set forth in its entirety.

The invention will be more fully understood upon consideration of thefollowing non-limiting examples.

EXAMPLES

Methods

Mutagenesis and Characterization of CHO-K1 Cells

A mutant cell line lacking the receptor was generated, so that thisdefect could be genetically complemented. About 5×10⁷ cells of thehypodiploid CHO-K1 cell line were treated at 37° C. for 7 hr with mediumcontaining 10 μg/ml ICR-191 (Sigma), a DNA alkylating agent that inducessmall deletions and frameshift mutations in genes, then washed twice.This treatment led to approximately 90% cell death.

The surviving mutagenized cells were then challenged with 8 μg/ml PA and10 ng/ml LF_(N)-DTA, a fusion protein composed of the N-terminal 255amino acids of LF linked to the catalytic A chain of diphtheria toxin.This recombinant toxin can kill CHO-K1 cells (in contrast to LF and PA)and it exploits the same LF/PA/receptor interactions that are requiredfor the binding and entry of the native LF and EF proteins. After 4days, surviving cells were replated and incubated for 3 days with mediumcontaining PA and LF_(N)-DTA. Ten single-cell colonies (designated asCHO-R1.1 to CHO-R1.10) that survived toxin treatment were isolated 14days later. In control experiments performed with non-mutagenized CHO-K1cells, no toxin-resistant cell clones were detected.

One of the mutagenized clones (CHO-R1.1) was chosen for furtheranalysis. CHO-R1.1 cells were found to be fully susceptible to killingby diphtheria toxin (DT) by measuring ³H-leucine incorporation intocellular proteins after exposure to the toxin, thus ruling out thepossibility that resistance to PA/LF_(N)-DTA was due to a defect in thepathway of DT action. To test directly whether CHO-R1.1 cells lacked thereceptor, flow cytometric analysis was performed after the cells wereincubated at 4° C. for 2 hr in medium containing 40 to 80 nM PA-K563Ccoupled at mutated residue 563 to Oregon Green maleimide (MolecularProbes) (“OGPA”). The treated cells were washed twice with medium andanalysed using a Becton Dickinson FACSCalibur flow cytometer. CHO-R1.1cells were significantly impaired in their ability to bind to OGPA ascompared to the parental cell line, suggesting that these mutagenizedcells had lost expression of the putative PA receptor gene. Similaranalysis of the other nine mutant CHO-R1 clones demonstrated that theywere also defective in binding to OGPA.

cDNA Complementation

In an attempt to complement the PA binding defect of CHO-R1.1 cells, thecells were transduced with a retrovirus-based cDNA library (Clontech)prepared from human HeLa cells that express the PA receptor. This cDNAlibrary is contained in a murine leukemia virus (MLV) vector that ispackaged into pseudotyped virus particles (MLV[VSV-G]) containing thebroad host-range G protein of vesicular stomatitis virus (VSV-G).Retrovirus-based cDNA libraries are useful for genetic complementationapproaches since they can deliver a limited number of stably expressedcDNA molecules per cell. These molecules can be rapidly re-isolated byPCR amplification using MLV vector-specific oligonucleotide primers.

Approximately 5×10⁵ CHO-R1.1 cells were transduced with about 10⁷infectious units (complexity of library=2×10⁶ independent clones) of thepLIB-based cDNA library (Clontech; cat.# HL8002BB) produced in the293GPG packaging cell line. Three days later, cells were incubated withmedium containing 80 nM OGPA and the top 0.1% of fluorescent cells werethen isolated by sorting using a Becton Dickinson FACSVantageSEinstrument. Cells were sorted based on their binding of OGPA incombination with an anti-PA polyclonal serum and an allophycocyanin(APC) conjugated secondary antibody. To isolate those that contained theputative PA receptor cDNA clone, these cells were expanded and subjectedto four additional rounds of sorting using OGPA as above, as well as a1:500 dilution of a rabbit anti-PA polyclonal serum along with a 1:500dilution of an APC-conjugated secondary antibody (Molecular probes).OGPA-single positive (round 2) or OGPA/APC-double positive (rounds 3–5)cells were recovered (the top 20%, 1%, 5%, and 50% of fluorescent cellsfor rounds 2, 3, 4, and 5 respectively) and expanded after each round ofsorting.

This led to the isolation of a cell population in which greater than 90%of the cells bound OGPA. This complemented cell population contained atleast seven unique cDNA inserts that were obtained by the PCRamplification method described above. Each cDNA was gel purified,subcloned back into the parent pLIB vector and packaged into MLV(VSV-G)virions so that it could be tested for its ability to complement thePA-binding defect of CHO-R1.1 cells. One cDNA clone of approximately 1.5kb (designated as ATR) restored PA binding to CHO-R1.1 cells. This clonealso dramatically enhanced the binding of PA to parental CHO-K1 cells.

Furthermore, the ATR cDNA clone fully restored LF_(N)-DTA/PA toxinsensitivity to CHO-R1.1 cells. In this test, CHO-R1.1 cells and CHO-K1cells were either not transduced or transduced with the MLV vectorencoding ATR; these cells were treated with 10⁻⁹ M LF_(N)-DTA andvarious concentrations of PA; medium containing 1 μCi/mL ³H-leucine wasthen added to cells for 1 hr, and the amount of ³H-leucine incorporatedinto cellular proteins was determined by trichloroacetic acidprecipitation and liquid scintillation counting.

cDNA Characterization

cDNA inserts were recovered from these cells by PCR amplification ofgenomic DNA samples using oligonucleotide primers specific for the MLVvector according to the manufacturers instructions (Clontech). Each cDNAwas subcloned between the NotI and SalI restriction enzyme sites of pLIBand the resulting plasmids were co-transfected into 293 cells with MLVgag/pol and VSV-G expression plasmids pMD.old.gagpol and pMD.G.Resulting pseudotyped virus particles were used to infect CHO-R1.1 andCHO-K1 cells followed by OGPA staining and FACS analysis as above.

Sequencing of the ATR cDNA clone revealed a single long open readingframe, encoding a 368 amino acid protein. FIG. 1 shows sequencealignment of ATR (SEQ ID NO:2) with the von Willebrand factor A domainconsensus sequence (SEQ ID NO:3; VWA-CON), the I domain of integrin α2(SEQ ID NO:4; α2), and TEM8 (SEQ ID NO:6). The secondary structuralelements are based on the crystal structure of the α2 I domain.Conserved amino acids are boxed and identical amino acids are indicatedby shaded boxes. The putative signal sequence is underlined. The fiveresidues that form the MIDAS motif are indicated with asterisks. Theputative transmembrane domains of ATR and TEM8 are indicated with ashaded box. Potential N-linked glycosylation sites in ATR and TEM8 areindicated by hatched boxes. The alignment was made using the programsClustalW and ESPript 1.9.

The ATR protein is predicted to have a 27 amino acid long signalpeptide, a 293 amino acid long extracellular domain with three putativeN-linked glycosylation sites, a 23 amino acid long putativetransmembrane region, and a short cytoplasmic tail. A BLAST searchrevealed that the first 364 amino acids of ATR are identical to aprotein encoded by the human TEM8 cDNA clone (Genbank accession numberNM 032208). The C-terminal ends of ATR and the TEM8 protein thendiverge, presumably as a consequence of alternative splicing, such thatATR has a cytoplasmic tail of only 25 amino acids whereas TEM8 ispredicted to have a 221 amino acid long cytoplasmic tail. The mostnotable feature of ATR is the presence of an extracellular vonWillebrand Factor type A (VWA) domain, located between residues 44 and216.

The cytoplasmic tail of ATR contains an acidic cluster (AC motif)(EESEE) that is similar to a motif found in the cytoplasmic tail offurin which specifies basolateral sorting of this protease in polarizedepithelial cells. This may be significant because the PA receptorlocalizes to the basolateral surface of polarized epithelial cells andit is expected that the receptor and the protease needed to bind andactivate PA would be co-localized to allow for efficient entry ofanthrax toxins.

Cloning and Expression of T7-ATR₄₁₋₂₂₇

A fusion protein having a hexahistidine tag, a T7 tag, and amino acids41 to 227 of ATR (the I domain) was constructed, expressed and purifiedfrom E. coli cells as follows. A DNA fragment encoding amino acids41–227 of ATR was cloned into the BamH1 and EcoR1 sites of pET28A(Novagen) to generate pET28A-ATR₄₁₋₂₂₇. BL21 (DE3) cells (Stratagene)containing pET28A-ATR₄₁₋₂₂₇ were grown at 37° C. to an OD₆₀₀ of 0.6,induced with 1 mM isopropyl-β-D-thiogalactopyranoside for 4 hr andharvested by centrifugation. The cells from 1.5 L of culture wereresuspended in 25 mL of 50 mM Tris-HCl pH 8.0, 2 mM dithiothreitol(DTT), 1 mM phenylmethylsulfonyl fluoride and were passed through aFrench press. One milligram of DNAse I (Roche) was added to the celllysate, which was then sonicated for 1 min and centrifuged at 21,000 gfor 20 min. The pellet was resuspended in 25 mL of 50 mM Tris-HCl pH8.0, 2 mM DTT and centrifuged at 21,000 g for 20 min. This wash step wasrepeated once. T7-ATR₄₁₋₂₂₇ was solubilized and folded essentially asdescribed previously.

When mixed with wild-type PA (on ice for 30 min), this construct wasprecipitated with polyclonal anti-PA serum (analyzed by SDS-PAGE andWestern blot using anti-T7 antibody conjugated to horseradishperoxidase). The interaction between PA and T7-ATR₄₁₋₂₂₇ was impaired bythe presence of EDTA (2 mM), demonstrating that the involvement ofdivalent cations in the interaction, and suggesting that the ATR MIDASmotif is involved in binding PA.

Interaction Between PA and ATR

PA-N682S, a mutant form of PA isolated as described below and having animpaired ability to bind and intoxicate cells, did not bind toT7-ATR₄₁₋₂₂₇. The DNA encoding Domain 4 of PA was mutagenized usingerror-prone PCR. Clones were expressed in E. coli, and lysates derivedfrom these clones were added to CHO-K1 cells in combination withLF_(N)-DTA. Clones corresponding to lysates that did not kill CHO-K1cells were sequenced and the N682S mutant clone was furthercharacterized as having Ser in place of Asn at position 682.

PA-N682S was shown to have an impaired ability to bind cells as follows.CHO-K1 cells were incubated with 2×10⁸ M trypsin-nicked PA (wild-type orN682S) for 1 hr, washed with PBS, resuspended in SDS sample buffer andrun on a 4–20% polyacrylamide SDS gel, and PA was visualized by Westernblotting. In the experiment in which PA-N682S was shown to have animpaired ability to intoxicate cells, CHO-K1 cells were incubated withLF_(N)-DTA (10⁻⁹ M) and various concentrations of wild-type PA orPA-N682S mutant, and cell viability was determined.

To confirm that PA binds directly to ATR, co-immunoprecipitations (usinga polyclonal serum specific for PA and protein A agarose) were performedwith an extracellular fragment of ATR and either the wild-type or areceptor binding-deficient mutant form of PA. A mixture of 5 μg PA (WTor N682S) and 2 μg T7-ATR₄₁₋₂₂₇ (in 20 mM Tris-HCl pH 8.0, 150 mM NaCl,0.1 mg bovine serum albumin per mL) was incubated on ice for 30 min inthe presence or absence of 2 mM EDTA. Anti-PA polyclonal serum (10 μL)was added to this solution and incubated on ice for an additional 1 hr.Protein A agarose (Santa Cruz Biotechnology) was added and the solutionwas rotated at 4° C. for 1 hr, then washed four times with 20 mMTris-HCl pH 8.0, 150 mM NaCl. Approximately one third of the mixture wassubjected to SDS-PAGE, transferred to nitrocellulose and probed withanti-T7 antibody conjugated to horseradish peroxidase (Novagen).

In addition, a fusion protein containing GST and the PA receptor-bindingdomain (D4) (GST-D4) bound T7-ATR₄₁₋₂₂₇, while GST did not. DNA encodingamino acids 595 to 735 of PA (domain 4) was cloned into pGEX-4T-1(Pharmacia Biotechnology). This vector encoded the GST-D4 fusionprotein. GST-D4 was coupled to glutathione sepharose at a concentrationof 4 mg GST-D4 per mL according to manufacturer's instructions(Pharmacia Biotechnology). GST or GST-D4 coupled to glutathionesepharose was mixed with 2 μg of T7-ATR₄₁₋₂₂₇ and 250 μg of E. coliextract in a volume of 250 μL for 1 hr at 4° C. The beads were washed 4times with 20 mM Tris-HCl pH 8.0, 150 mM NaCl. One half of thesuspension was subjected to SDS-PAGE, transferred to nitrocellulose, andprobed with anti-T7 antibody coupled to horseradish peroxidase.

Taken together, the experiments described above demonstrate a direct andspecific interaction between the VWA/I domain of ATR and thereceptor-binding domain of PA. Given this direct interaction, wereasoned that ATR₄₁₋₂₂₇ might protect CHO-K1 cells from killing by PAand LF_(N)-DTA. This idea was tested by incubating (37° C. for 4 hr)CHO-K1 cells with an increasing amount of T7-ATR₄₁₋₂₂₇ in the presenceof a constant amount of PA (10⁻¹⁰ M)/LF_(N)-DTA (2.5×10⁻¹¹ M), and thenmeasuring the subsequent effect on protein synthesis. T7-ATR₄₁₋₂₂₇ wasan effective inhibitor of toxin action, inhibiting toxin activity by 50%and 100% at concentrations of 80 nM and 500 nM respectively.T7-ATR₄₁₋₂₂₇ did not, however, inhibit diphtheria toxin.

The present invention is not intended to be limited to the foregoing,but encompasses all such modifications and variations as come within thescope of the appended claims.

1. An isolated polynucleotide or complement thereof, the polynucleotidecomprising a nucleotide sequence encoding the amino acid sequence of SEQID NO:2.
 2. An isolated polynucleotide or complement thereof, thepolynucleotide encoding an the amino acid sequence selected from thegroup consisting of SEQ ID NO:2, amino acids 27–321 of SEQ ID NO:2, andamino acids 28–320 of SEQ ID NO:2.
 3. The isolated polynucleotide ofclaim 1 comprising SEQ ID NO:1 from position 104 to 1207 or thecomplement thereof.
 4. An isolated polynucleotide or complement thereof,the polynucleotide encoding the amino acid sequence selected from thegroup consisting of amino acids 41–227 of SEQ ID NO:2, amino acids42–222 of SEQ ID NO:2, and amino acids 44–216 of SEQ ID NO:2.
 5. Theisolated polynucleotide of claim 4 wherein the polynucleotide encodes anthe amino acid sequence selected from the group consisting of aminoacids 41–227 of SEQ ID NO:2 and amino acids 42–222 of SEQ ID NO:2.
 6. Avector comprising the polynucleotide of claim
 1. 7. A vector comprisinga non-native expression control sequence operably linked to apolynucleotide selected from the group consisting of the polynucleotideof claim 1 and a polynucleotide of claim
 4. 8. The vector of claim 7,wherein the polynucleotide is selected from the group consisting of thepolynucleotide of claim 1 and a polynucleotide of claim
 4. 9. A hostcell comprising a non-native expression control sequence operably linkedto a polynucleotide selected from the group consisting of thepolynucleotide of claim 1 and a polynucleotide of claim
 4. 10. The hostcell of claim 9, wherein the polynucleotide is selected from the groupconsisting of the polynucleotide of claim 1 and a polynucleotide ofclaim
 4. 11. A method for producing an anthrax toxin receptor, themethod comprising the steps of: transcribing a polynucleotide operablylinked to an upstream expression control sequence, wherein thepolynucleotide is selected from the group consisting of thepolynucleotide of claim 1 and a polynucleotide of claim 4 to produce anmRNA; and translating the mRNA to produce the anthrax toxin receptor.12. A method as claimed in claim 11, wherein the polynucleotide isoperably linked to the expression control sequence in an expressionvector, and wherein the expression vector is delivered into a host cell,the expression control sequence being operable in the host cell.
 13. Amethod as claimed in claim 11, wherein at least one of the transcribingand translating steps are performed in vitro.
 14. The method of claim11, wherein the polynucleotide is selected from the group consisting ofthe polynucleotide of claim 1 and a polynucleotide of claim 4.